****************************************************************************** From: "Orval A. Mamer" Date: Tue, 6 Oct 1998 11:54:38 -0400 Subject: Calibration with water clusters Organization: McGill University Computing Centre Dear Group, I would like to bring to the attention of everyone an alternative to PEG and CsI for calibration of quadrupoles in electrospray mode. From discussions with my fellow mass spectrometrists, it seems that at this moment, most scientists performing electrospray analysis are using either PEG, CsI or NaI as a calibrant. A disadvantage for using PEG is if you want to calibrate up to 3000 Da, the C13 isotope ions get more and more intense and this can make things more difficult for the calibration software to pinpoint the proper C12 ions. To palliate this problem, we preferred to use the monoisotopic CsI clusters instead. For our applications, we were able to calibrate from 132 to 3700 Da in positive mode, and from 127 to 2000 Da in negative mode. Of course, nothing being perfect in this world, the CsI cluster signal intensities diminishes rapidly with increasing m/z values, which means that longer acquisition times have to be used to get a strong enough signal for the calibration software. Furthermore, CsI is a non volatile salt and will crystallize on the source surfaces impairing source performance, which means that after calibrating the quadrupoles, we had to clean the source. For all these reasons above, we decided to try the calibration method presented by Ledman and Fox in the Journal of the American Society for Mass Spectrometry (vol 8, 1997, pp 1158 to 1164). It worked like a charm! The secret is to favor water cluster formation by using a cool source, low drying gas flowrate and no organic solvent at all. Only pure water with 0.05% trifluoroacetic acid to induce protonation. All the masses of water clusters from 19 to 3062 Da are listed in Table 1 of the Ledman paper. So, it is very easy to update the computer reference files with the proper values. With this method, we were able to calibrate successfully and easily from 15 to 3067 Da on our Quattro II. We have found out that even on the full range spectrum from 195 to 3067 Da, it was still possible to see the ions above 2000 without magnification. And the most wonderful thing about using water is that it will not get the source dirty, au contraire! We are so pleased with this method, that we wanted to share our experience with all. Daniel Boismenu, Ph.D. Research Associate Biomedical Mass Spectrometry Unit McGill University 1130 Pine ave, West Montréal, Québec Canada H3A 1A3 Tel : (514) 398-3661 Fax : (514) 398-2488 mcdl@musica.mcgill.ca ****************************************************************************** From: A.P.Bruins (a.p.bruins@farm.rug.ml) Date: Wed, 7 Oct 1998 10:20:54 MET Subject: Re: Mass scale calibration on water clusters Organization: Pharmacy Dept Groningen University Mass scale calibration on water clusters It is amazing that there is suddenly strong interest in mass scale calibration on water clusters. I have used this method ever since I converted my NERMAG R 3010 to APCI and Electrospray in 1988 (see the ASMS 1988 proceedings). The paper by Ledman presents water clusters generated by electrospray. What Ledman and other authors overlook is the fact that you do not even need to use water and electrospray to make water clusters. All I have used during the past ten years is room air and a corona discharge, to make water clusters. The secret is to make use of the strong cooling of ions and room air (containing some moisture) in the free jet expansion into the vacuum system. What you need is the H3O+ ion and just the right amount of moisture to create the clusters. The right amount of moisture is found experimentally by turning the curtain gas (Sciex-type ion sources as in the API 100/300/3000 series) almost completely off, so that water can associate with the H3O+ ions. If you turn the curtain gas off completely, you will see no ions at all, since the size of the clusters is far too big. So you have to tune for the best cluster ion abundance by varying the curtain gas flow. Since there may be some ammonia present in room air, you may find two series of cluster ions, one with NH4+ as the core ion, and the other one with H3O+ as the core ion. This of course may create a 1 m/z calibration error, and can easily be overcome by putting an open bottle of aqueous ammonia solution close to the ion source, and draw the head space of this bottle through the APCI source. In this way the H3O+ clusters will disappear, and you'll only get clusters with NH4+ as the core ion. Also make shure that there are no open bottles of HPLC eluents in the room, to avoid the formation of clusters containing methanol or acetonitrile. Good luck! For the sake of completeness, here is the text on calibration I wrote for the ESI book edited by R. Cole, see page 107: Calibration of the mass scale requires a series of ions evenly spaced throughout the mass range. Ideally, the calibrant should be easily removed from the source and leave no traces in the source or liquid handling system. Mixtures of a protein (usually myoglobin) and some smaller peptides can be used; polyethylene glycols and polypropylene glycols are also widely used for calibration. Anacleto et al. have summarized different options, and have proposed protonated water clusters and salt clusters (39), generated by pneumatically assisted electrospray. Water clusters provided a calibration range up to m/z 1000 in the SCIEX API III mass spectrometer. Alkali metal halides (sodium iodide) allow calibration on cluster ions Na+(NaI)n or I-(NaI)n up to m/z 2400, the full mass range of this instrument. In principle, mass scale calibration is independent from the ionization technique. During the past seven years we have routinely calibrated by the use of a combination of corona discharge and controlled cluster formation in the free jet expansion. Corona discharge of the headspeace of an aqueous ammonia solution creates NH4+(H2O)n with n=0-4 after passage through the gas curtain. If the curtain gas flow is reduced but not cut off one can tune the gas flow to allow a controlled amount of moisture into the free jet expansion so that a complete series of NH4+(H2O)n ions is formed that extends all the way up to m/z 2000 (mass range of a NERMAG R3010) and most probably beyond this value. The calibration mass series starts at m/z 54, and one out of every five clusters is used, giving a calibration mass separation of 90 amu. The relatively low abundance of deuterium and 17 O, and the higher abundance of 18 O in the cluster ions makes this calibrant suitable for resolution adjustment on isotope peaks. Andries Bruins Dr. A.P. Bruins University Centre for Pharmacy A. Deusinglaan 1 9713 AV Groningen, The Netherlands phone +31-50-363-3262 fax +31-50-363-3311 a.p.bruins@farm.rug.nl